Introduction

In contrast to the sexual reproduction of themycobiont which requires re-lichenization with asuitable photobiont, the asexual reproduction bysoredia, isidia or schizidia are regarded as idealadaptations to lichens which are composed offungal and algal partners. Unfortunately, howev-er, we have only a limited knowledge on the de-velopment of these vegetative propagules intothalli either in the field or under culture. Schei-degger (1995) reported early development ofisidioid soredia in Lobaria pulmonaria andSchuster et al. (1985) also reported early growthand development of soredia for Hypogymniaphysodes and Physcia tenella in the natural envi-ronment. Stocker-Wörgötter & Türk (1988) alsosucceeded producing young thalli from sorediaof Peltigera didactyla. However, development oftrue isidia has been observed only in Peltigerapraetextata, a cyanobacterial lichen (Stocker-Wörgötter & Türk, 1995).

Parmotrema tinctorum (Nyl.) Hale is a com-mon foliose lichen containing Trebouxia (a greenalga) as the photobiont. It is widely distributed in

temperate and tropical areas in the world, beingeasily recognized by the e-ciliate rounded lobeswith dense isidia and by the distinct C� red reac-tion in the medulla. It has the potential for twodivergent strategies of reproduction, i.e. sexual,through ascospores ejection, and vegetative bythe symbiotic propagules, isidia. However, fertilespecimens are found only rarely in luxuriant pop-ulations and they are absent in most localities inJapan. Therefore, dispersal by ascospores mayplay only limited role in reproduction in mostpopulations. In contrast, isidia are regularlyfound even in small thalli in small populationsand may play major role in dispersal.

In the present paper, the successful develop-ment of juvenile lobules from isidia will be re-ported for Parmotrema tinctorum. The structureof both young and mature isidia was also ob-served.

Materials and Methods

Isidia used for the present study were collectedfrom Parmotrema tinctorum growing on thetrunks of trees at Mt. Kiyosumi, Chiba Prefec-

Lobule Formation from Isidia in Parmotrema tinctorum

Yoshiaki Kon1 and Hiroyuki Kashiwadani2

1 Kohoku Senior High School, 4–14–30 Nishiayase, Adachiku, Tokyo, 120–0014 Japan

E-mail: yoshkon@xw.catv.ne.jp2 Department of Botany, National Science Museum,

4–1–1 Amakubo, Tsukuba, 305–0005 JapanE-mail: hkashiwa@kahaku.go.jp

Abstract Vegetative diaspores (isidia) of Parmotrema tinctorum were kept in nylon bags. Thesenylon bags were stapled on the trunks of Cryptomeria japonica. Morphological changes of isidiawere observed every three months after stapling in the natural field. Several protuberances wereformed on the isidia six months after stapling. Isidia with protuberances at this stage were an-chored by thin bundles of hyphae to the nylon bags. These protuberances developed into dorsiven-tral lobules with rhizines 12 months after stapling and the lobules were stratified as seen in the nat-ural thalli of P. tinctorum. Lecanoric acid and atranorin were produced in the newly developed lob-ules.Key words : Isidia, lobules formation, Parmotrema tinctorum.

Bull. Natn. Sci. Mus., Tokyo, Ser. B, 31(4), pp. 127–131, December 22, 2005

ture. They were cut off with a knife from thalliand kept in bags about 5�5 cm in size whichwere made of 150 mm meshed nylon. Each bagcontained about 30 cylindrical or branched isidia(ca. 100–150 mm in length). These nylon bagswere stapled on the trunks of Cryptomeria japon-ica D. Don growing near by the collecting site(Fig. 1). Developmental stages of isidia were in-vestigated by light microscopy and scanningelectron microscopy every three months after sta-pling. For light microscopic observation, sectionswere made by using a freezing microtome andwashed with acetone solution in order to removethe secondary substances such as lecanoric acidand atranorin. Naturally dried specimens werecemented on specimen-holders, sputter coatedwith about 100 Å gold, and examined at 15 kV ina JEOL JSM-5410LV scanning electron micro-scope. Lobules newly formed on the isidia werechemically tested by the standardized thin-layerchromatographic method (Culberson, 1972).

Results and Discussion

Formation of isidiaIn the initial stage, isidia appear to be small

verrucae on the upper surface in P. tinctorum. In

this stage (Fig. 2B), the verrucae are filled withalgal cells, which are mostly the same as those ofthe medulla in size and have cortex extendedfrom the upper cortex of the thallus. As isidiagrow a little further (Fig. 2C) and become shortcylindrical projections, algal cells increase innumber. It is noteworthy that algal cells formedinside of the isidial projections are smaller in sizecomparing those derived from or in the thallus.In the later stages, these projections become dis-tinct cylindrical isidia (Fig. 2D), which can berecognized even with the naked eye. In this stage,algal cells are concentrically distributed belowthe cortex and medullary hyphae of the thalluspenetrate into the central part of isidia (Fig. 2A).However, no distinct stratification is observed.

Formation of juvenile thalli from isidiaNylon bags staled on the trunks of Cryptome-

ria japonica contained about 30 isidia in variousstages of development. However they were most-ly cylindrical or coralloid. In the present study,cylindrical isidia were mainly observed. Cylin-drical isidia were about 75 mm in diameter and100–200 mm long (Fig. 3A).

The cut end of isidia was exposed when thenylon bags were stapled (Fig. 3A). Three months

128 Yoshiaki Kon and Hiroyuki Kashiwadani

Fig. 1. Nylon bag stapled on the trunks of Cryptomeria japonica. Scale�10 mm.

after stapling, it was covered by mycobiont hy-phae which seemed to adhere each other withgelatinous substances (Fig. 3B). In the presentstudy, isidia of P. tinctorum did not seem to befixed on 150 mm nylon mesh.

Six months after stapling, a clear morphologi-cal changes of isidia were observed. In this stage,each isidium produces several verrucae or protu-berances 20–30 mm broad and 50–60 mm long(Fig. 3C). There is no distinct boundary betweenisidia and verrucae or protuberances and cortexof isidia is spontaneously joined to those of ver-rucae or protuberances. On some of them, bun-dles of hyphae tightly anchoring to the substra-tum (nylon fiber) were observed (Fig. 3C). Theyare comparable with anchoring hyphae reportedin the early stages of development of isidioid

soredia of Lobaria pulmonaria by Scheidegger(1995).

Twelve months after stapling, protuberancesdeveloped into dorsiventral lobules, which werepale gray and 0.2–0.35 mm broad and 0.2–0.3 mm long (Fig. 3D).

The cortex was often disintegrating at thebasal parts of lobules as well as on the isidia(Fig. 3D). In this stage distinct rhizines were ob-served on the lower surface of the lobules (Fig.3E). At present, however, it is uncertain whetherthese rhizines have derived directly from anchor-ing hyphae mentioned above or not. It should benoted here that lobules in this stage were clearlystratified, upper and lower cortices, algal layerand medulla being recognized (Fig. 3F), beingquite similar to thalli of P. tinctorum in nature,

Lobule formation from isidia in Parmotrema tinctorum 129

Fig. 2. Inner structure of isidia of Parmotrema tinctorum. A: Cross-section of mature isidia formed on the thalliof P. tinctorum. Algae are distributed concentrically under the cortex of the isidia and hyphae are situated inthe center of isidia. B–D: Longitudinal section of young and mature isidia of P. tinctorum. B: Juvenile isidia.C: Young isidia. D: Mature isidia. (A–C scales�50 mm, D scale�100 mm).

though the medulla was rather thin. In addition,secondary products of newly formed lobuleswere tested by the TLC method and lecanoricacid and atranorin, constant components in P.

tinctorum, were demonstrated on the chromato-grams.

Stocker-Wörgötter & Türk (1995) observedthat isidia transformed into soredial primordial at

130 Yoshiaki Kon and Hiroyuki Kashiwadani

Fig. 3. Scanning electron micrography of developmental stages of lobule formation observed in the field and alobule. A: Cylindrical isidium removed from a thallus. B: Isidium three months after stapling. The cut end(arrow) is covered with hyphae. C: Verrucae and protuberances formed on the isidia six months after sta-pling, showing bundles of hyphae anchoring to the substratum (arrow). D: Lobules formed on the isidia 12months after stapling. E: Lobules 12 months after stapling, showing rhizines (arrow) on the lower surface. F:Cross-section of lobules formed on isidia 12 months after stapling. Stratified composition is similar to that oforiginal thalli of P. tinctorum, though the medulla is rather thin. Scale�100 mm.

first and then small lobules were differentiatedfrom fused soredial clumps in Peltigera praetex-tata. In P. tinctorum, however, such developmen-tal process was not observed in any stages andverrucae or protuberances were formed directlyon isidia and they developed into small lobules.When Scheidegger (1995) investigated early de-velopment of isidioid soredia of Lobaria pul-monaria, he reported that apical and lateralpseudomeristematic growth zones were formedsix months after transplantation and then thegrowth zones further differentiated into smalllobes which have a stratified thallus as seen in fo-liose lichens. In P. tinctorum, however, verrucaeor protuberances were formed in the early stagesand they directly developed into lobules. Thus,verrucae or protuberances can be considered tobe equivalent to pseudomeristems observed byScheidegger (1995). It is noteworthy, on the otherhand, these verrucae or protuberances resemblevery much those observed on the periphery ofthe transplanted fragments of P. tinctorum (Konet al., 2003).

Isidia of P. tinctorum are cylindrical and algalcells are concentrically distributed inside of thecortex and protuberances formed on isidia seemto have a similar inner morphology as isidia. Incontrast, lobules developed from isidia are clear-ly stratified as mention above, though the medul-la is rather thin (Fig. 3F). Therefore, dispositionof algal cells and fungal hyphae should bechanged, while dorsiventral lobules are develop-ing, although such process has not been observedin the present study.

It is now obvious that isidia removed from thethallus commonly produce many protuberancesand juvenile lobules in six to twelve months after

detaching from the thallus. As having been sus-pected by most lichenologists, thus, isidia can beconsidered to play a major and important role indispersal in P. tinctorum. Since regeneration ofisidia has never been observed while they adhereonto the thallus, formation of verrucae or protu-berances on isidia observed through the presentstudy seems to be triggered by detaching fromthe thallus.

Acknowledgements

We express our sincere thanks to Dr. S.Kurokawa, Botanical Gardens of Toyama for hisvaluable suggestion and criticism.

References

Culberson, C. F., 1972. Improved conditions and data forthe identification of lichen products by standardizedthin-layer chromatographic method. J. Chromatogr., 72:113–125.

Kon, Y., M. Mineta & H. Kashiwadani, 2003. Transplan-tation experiment of lichen thalli of Parmotrema tincto-rum (Ascomycotina, Parmeliaceae). J. Jpn. Bot., 78:208–213.

Scheidegger, C., 1995. Early development of transplantedisidioid soredia of Lobaria pulmonaria in an endan-gered pollution. Lichenologist, 27(5): 361–374.

Schuster, G., S. Ott and H. M. Jahns, 1985. Artificial cul-ture of lichen in the natural environment. Lichnologist,17(3): 247–253.

Stocker-Wörgötter, E. and R. Türk, 1988. Culture of thecyanobacterial lichen Peltigera didactyla from sorediaunder laboratory conditions. Lichenologist, 20(4):369–375.

Stocker-Wörgötter, E. and R. Türk, 1995. Experimentalcultivation of lichen and lichen symbionts. C. J. Bot.,73 (Suppl. 1): s579–s589.

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